NCL Method ITA-5.2 May 2020 1 Version 3
NCL Method ITA-5.2
Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA
Nanotechnology Characterization Laboratory Frederick National Laboratory for Cancer Research
Leidos Biomedical Research, Inc. Frederick, MD 21702
(301) 846-6939 [email protected]
https://ncl.cancer.gov
NCL Method ITA-5.2 May 2020 2 Version 3
Method written by:
Barry W. Neun
Edward Cedrone
Marina A. Dobrovolskaia*
Nanotechnology Characterization Lab, Cancer Research Technology Program, Frederick
National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick,
MD 21702
*- address correspondence to: [email protected]
Protocol adapted from:
Neun BW, Ilinskaya AN, Dobrovolskaia MA. Analysis of Complement Activation by
Nanoparticles, in Characterization of nanoparticles intended for drug delivery, S. McNeil,
Editor. Methods in Molecular Biology. Vol. 1628, 2018, Humana Press, New York, NY. p. 149-
160. doi: 10.1007/978-1-4939-7352-1_13
Please cite this protocol as:
Neun B, Cedrone E, Dobrovolskaia MA, NCL Method ITA-5.2: Analysis of Complement
Activation by single-plex EIA or multiplex ELISA. https://ncl.cancer.gov/resources/assay-
cascade-protocols DOI: 10.17917/EQCT-3C51
NCL Method ITA-5.2 May 2020 3 Version 3
1. Introduction
This document describes a protocol for quantitative determination of complement activation
by an Enzyme Immunoassay (EIA). The complement system represents an innate arm of immune
defense and is named so because it “complements” the antibody-mediated immune response.
Three major pathways leading to complement activation have been described: they are the
classical pathway, alternative pathway and lectin pathway (Figure 1). The classical pathway is
activated by immune (antigen-antibody) complexes. Activation of the alternative pathway is
antibody independent. The lectin pathway is initiated by plasma protein mannose-binding lectin.
The complement system is a group of ~30 protein that includes several components (C1 -
C9), and Factors (B, D, H, I, and P). Activation of any of the three pathways results in cleavage
of the C3 component of the complement system [1, 2].
This protocol is intended for follow-up studies on samples which demonstrated a positive
response in the qualitative assay (NCL Method ITA 5.1). It can also be performed as an
independent protocol when high throughput analysis is needed.
2. Principles
In the protocol presented herein, human plasma is exposed to a test material and
subsequently analyzed by EIA for the presence of the complement components C4d, iC3b and
Bb. The antibodies specific to these proteins are immobilized on 96 well plates and are obtained
from commercial suppliers. Test nanoparticles found to be positive in the qualitative western blot
assay are then subject to a more detailed investigation aimed at delineation of the specific
complement activation pathway. Detection of elevated levels of C4d protein is indicative of
complement activation via the classical or lectin pathway. Elevation in Bb levels is a sign of
alternative pathway activation. Estimation of iC3b levels is used to confirm, in a more accurate,
quantitative way, the results of the initial western blot screen specific to the C3 component of the
complement system.
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Figure 1. Complement activation pathways. (This illustration is reproduced from
reference 1 with permission from EMD Biosciences, Inc.)
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3. Reagents, Materials, and Equipment
Note: The NCL does not endorse any of the suppliers listed below; these reagents were used
in the development of the protocol and their inclusion is for informational purposes only.
Equivalent supplies from alternate vendors can be substituted. Please note that suppliers
may undergo a name change due to a variety of factors. Brands and part numbers typically
remain consistent but may also change over time.
3.1 Reagents
3.1.1 Sterile Ca2+/Mg2+-free phosphate buffered saline (PBS) (GE Life Sciences,
SH30256.01)
3.1.2 Cobra Venom Factor (positive control) (Quidel Corp., A600)
3.1.3 Veronal Buffer (Boston BioProducts, IBB-260)
3.1.4 Pooled human plasma, anti-coagulated with Sodium citrate
3.1.5 MicroVue iC3b EIA kit (Quidel Corp., A006)
3.1.6 MicroVue C4d fragment EIA kit (Quidel Corp., A0008)
3.1.7 MicroVue Bb Plus EIA kit (Quidel Corp., A027)
3.1.8 MicroVue Complement Multiplex (8-plex) EIA Kit (Quidel Corp., A900)
3.1.9 Doxil (Doxorubicin HCl, liposome, injection) This is a prescription
medication available from a licensed pharmacy and may not be available
to some research laboratories.
3.1.10 Cremophor, (Sigma, C 5135)
3.1.11 Complement activator (Quidel,
https://www.quidel.com/research/complement-reagents/complement-
activator)
3.1.12 Taxol (Paclitaxel in Cremophor EL) This is a prescription medication
available from a licensed pharmacy and may not be available to some
research laboratories.
3.2 Materials
3.2.1 Pipettes covering the range from 0.05 to 1 mL
3.2.2 Microcentrifuge tubes, 1.5 mL
3.2.3 Pipet tips, 0.5 µL – 1.0 mL
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3.2.4 Multichannel (8-12 channel) pipettor with volumes 50-300 µL
3.2.5 15 and 50 mL conical tubes
3.2.6 Reagent reservoirs
3.3 Equipment
3.3.1 Microcentrifuge
3.3.2 Centrifuge capable of running at 2500xg, with a swinging basket set up to
hold 5 cc vacutainer tubes
3.3.3 Refrigerator, 2-8˚C
3.3.4 Freezer, -20˚C
3.3.5 Vortex
3.3.6 Incubator, 37˚C
3.3.7 ELISA plate reader (for single plex) capable of operating at 405 nm
3.3.8 Q-View Imager Pro (for multi-plex) or similar imaging system
4. Reagent and Control Preparation
4.1 Positive Control 1 (Traditional substance known to activate complement)
4.1.1 Cobra Venom Factor (CVF) is supplied frozen solution. Thaw this stock,
prepare single use aliquots and store them at a nominal temperature of -80˚C for
as long as performance is acceptable. Avoid repeated freeze/thaw cycles. After
thawing single use aliquot and using it in the assay, discard any leftover material.
For this experiment, use 30 µL (1.1-50 U) of CVF solution. This control
activates complement system through alternative pathway.
4.1.2. Heat Aggregated Gamma Globulin (HAGG) acts similarly to naturally
occurring immune complexes and is very potent activator of complement through
the classical pathway. This control is available from Quidel under name
“Complement Activator”. Handling and storage are according to the
manufacturer’s instructions. Avoid repeated freeze/thaw cycles when stored at -20
°C.
4.2 Positive Control 2 (nanoparticle relevant)
4.2.1 Cremophor-EL
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Cremophor-EL is an excipient commonly used in the pharmaceutical
industry to dissolve hydrophobic drugs. Cremophor-EL is a nanosized
micelle which is known to induce complement activation related pseudo
allergy (CARPA) syndrome [2], and therefore is used as a nanoparticle
relevant control. The following procedure can be used to prepare
Cremophor-EL with a composition that is similar to the clinical
formulation of Paclitaxel (Taxol) (527 mg of purified Cremophor EL*
(polyoxyethylated castor oil) and 49.7% (v/v) dehydrated alcohol, USP
and 2 mg of citric acid per 1 mL). Store at room temperature.
To prepare Cremophor-EL, mix commercial Cremophor 1:1 with ethanol
containing 2 mg/mL of citric acid to mimic the concentration of
Cremophor-EL, citric acid, and ethanol used in Taxol and the generic
formulation of paclitaxel.
4.2.2 Cremophor-EL Formulated Paclitaxel (Taxol)
Taxol can be used as an alternative nanoparticle relevant positive control.
It is supplied at a stock concentration 6 mg/mL of paclitaxel. When used
in this assay, the final concentration of paclitaxel is 2 mg/mL. Store 2-8ºC.
4.2.3 PEGylated Liposomal Doxorubicin (Doxil)
Doxil can also be used as nanoparticle relevant positive control [3]. Doxil
is doxorubicin formulated in nanoliposomes. It is available through the
pharmacy as 20 mg of Doxorubicin HCl in 10 mL vehicle. Store 2-8ºC.
4.3 Inhibition/Enhancement Control (IEC)
Use positive control sample after the incubation. Prior to loading this sample onto
ELISA plate, add nanoparticles at the same final concentrations as in the study
samples. For example, one can mix 20 μL of the positive control sample and 10
μL of the test nanoparticle. The test result for this sample needs to be adjusted by
the dilution factor 1.5 prior to comparison of the test value to the test value of the
positive control sample. If the test results are different by no more than 25%, the
test nanoparticle at the given concentration does not interfere with detection of the
complement split product by ELISA.
4.4 Negative Control (PBS)
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Sterile Ca2+/Mg2+-free PBS is used as a negative control. Store at room
temperature for up to 6 months.
4.5 Vehicle Control (relevant to each given nanoparticle)
When nanoparticles are not formulated in saline or PBS, the vehicle sample
should be tested to estimate the effect of excipients on the complement system.
This control is specific to each given nanoparticle sample. It should be prepared to
match the formulation buffer of the nanoparticle by both composition and
concentration.
4.5 Stop Solution (HCl)
Stop solution is provided with each kit but can also be prepared separately. Dilute
stock hydrochloric acid to a final concentration of 1.0 N. Filter and store and room
temperature for up to 2 weeks.
5. Preparation of Study Samples
This assay requires 400 µL of nanoparticles in PBS at a concentration 3 times higher than the
highest final tested concentration. The concentration is selected based on the plasma
concentration of the nanoparticle at the intended therapeutic dose. For the purpose of this
protocol this concentration is called “theoretical plasma concentration”. Considerations for
estimating theoretical plasma concentration were reviewed elsewhere [4] and are summarized in
Box 1 below.
The assay will evaluate 4 concentrations: 10X (or when feasible 100X, 30X or 5X) of the
theoretical plasma concentration, theoretical plasma concentration and two 1:5 serial dilutions of
the theoretical plasma concentration. When the intended therapeutic concentration is unknown,
the highest final concentration is 1 mg/mL or the highest reasonably achievable concentration.
For example, if the final theoretical plasma concentration to be tested is 0.2 mg/mL, then a
stock of 6 mg/mL will be prepared and diluted 10-fold (0.6 mg/mL), followed by two 1:5 serial
dilutions (0.12 and 0.024 mg/mL). When 0.1 mL of each of these samples is added to the test
tube and mixed with 0.1 mL of plasma and 0.1 mL of veronal buffer, the final nanoparticle
concentrations tested in the assay are: 2.0, 0.2, 0.04 and 0.008 mg/mL.
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6. Plasma Collection and Storage
Blood is drawn into vacutainer tubes containing anticoagulant. Sodium citrate is ideal anti-
coagulant for this assay, however depending on phlebotomy paraphernalia, plasma anti-
coagulated with sodium citrate may result in high background in the ELISA assay. In this case
using K2EDTA as the anticoagulant is acceptable. The first 5-10 mL of blood should be
discarded and not used to prepare plasma. For optimal results, it is important to keep blood at
20-24ºC, to avoid exposure to high temperatures (summertime) and low temperatures
(wintertime), and to avoid prolonged (> 1 hr) storage. Blood is transported to the lab in a
contained Styrofoam box with warm packs (20-24°C). To prepare plasma, the blood is spun
down in a centrifuge 10 minutes at 2500xg. Plasma is evaluated for the presence of hemolysis.
Discolored plasma (an indication of hemolysis) is not used to prepare the pool. Individual plasma
specimens that did not show any indication of hemolysis are pooled and mixed in a conical tube.
Plasma must be used for complement testing within 1 hour after collection. Pooled plasma can be
used and prepared by mixing plasma from at least 2 individual donors. The assay can also be
performed in the plasma from individual donors. In this case analyze plasma from at least 3
donors.
It is possible to use pooled sodium citrate plasma from commercial suppliers, however, when
placing the order, one needs to notify the supplier that the plasma is intended for complement
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testing so no delays between blood draw and plasma collection occurs. The supplier then freezes
the plasma immediately after collection and ships it to the lab on dry ice. When using frozen
plasma for the complement activation assay, it is important to avoid repeated freeze/thaw cycles.
The frozen plasma should be thawed in a water bath containing ambient tap water, mixed gently
and used immediately after thawing. It is also advised to avoid indefinite storage of frozen
plasma at -20ºC. The sooner the frozen plasma is used, the better the results are. In general, the
degree of complement activation estimated by comparing intensity of the C3 split product in the
positive control with that of the negative control is greater in fresh plasma than in thawed
plasma.
7. Experimental Procedure for Sample Preparation
7.1 In a microcentrifuge tube, combine equal volumes (100 µL of each) of veronal
buffer, human plasma, and a test-sample (i.e., positive control, negative control,
nanoparticles, or vehicle control if different than PBS). Prepare two replicates of
each sample.
7.2 Vortex tubes to mix all reaction components, spin briefly in a microcentrifuge to
bring any drops down, and incubate in an incubator at a nominal temperature of
37˚C for 30 minutes.
7.3 Prepare 100 µL aliquots and either use in EIA immediately or freeze at -20ºC for
later analysis.
8. Experimental Procedure for Single-Plex EIA
8.1 Follow the manufacturer’s instructions to reconstitute complement standard, wash
buffers and controls.
8.2 Dilute plasma samples prepared in step 7.3 in complement specimen diluent
reagent (provided with each kit). Use the following dilution guide for each
individual assay:
iC3b – 1:1500 for positive control sample; 1:30 for negative control and other test
samples
C4d – 1:30 for all samples
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Bb – 1:75 for all samples
Note: The dilution factors should be determined by each laboratory and adjusted
if needed.
8.3 Follow manufacturer’s instructions for plate loading volumes, incubation time,
and plate washing.
8.4 Read plate on a plate reader at 405 nm.
9. Experimental Procedure for Multi-plex ELISA
9.1 Follow manufacturer’s instructions to reconstitute standards, wash buffer, and
controls.
9.2 Dilute plasma samples prepared in step 7.3 with sample diluent (provided with
each kit). PC, NC, and all samples are diluted 1:100.
9.3 Follow manufacturer’s instructions for loading volumes, incubation time, and
plate washing.
9.4 Capture a 300-second image of the plate on the Q-View Imager Pro or a 270-
second image on the Q-View Imager LS.
10. Data analysis
Do not forget to use the appropriate dilution factor for control and study samples. Compare
determined amount of complement components between positive control or study samples with
that in the negative control. An increase in the complement component species 2.0-fold or higher
above the background (negative control) constitutes a positive response. If a nanoparticle under
study generated a positive response in any of the EIA assays, compare the degree of activation
between this particle and the Doxil or other nanoparticle-relevant control. Doxil is used in the
clinic and is known to induce complement activation related hypersensitivity reactions in
sensitive patients [5]. Using Doxil helps to interpret results of this in vitro study for a test
nanoparticle. If the degree of activation observed for the test nanoparticle is equal to or greater
than that observed for Doxil, this nanoparticle formulation will most likely cause similar or
stronger hypersensitivity reactions in patients and may require modifications before entering in
vivo preclinical and clinical phases. If the degree of activation is lower than that of Doxil,
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complement activation should be considered when designing the in vivo evaluation phase for the
given particle, but it is less likely to cause concerns similar to Doxil.
11. Acceptance Criteria
11.1 Percent CV between replicates of standard curve, quality controls, and test
samples should be within 25%.
11.2. Percent difference from theoretical for each of the standard curve samples should
be within 25%, and correlation coefficient should be at or above 0.98.
11.3 Run is acceptable if conditions described in 11.1 and 11.2 are met.
11.4 The degree of complement activation in the positive control sample, estimated by
comparing levels of individual complement split product in the positive control
with that in the negative controls should be at or above 2.0-fold.
Note: Cobra venom factor activates complement through the alternative assay, so
this control will not provide a positive response in the C4d assay. HAGG is a
positive control for C4d assay. Doxil is positive in the C4d EIA.
12. References
1. The Complement System. Complement reagents of the highest quality. EMD
Biosciences, Calbiochem, Page 2.
http://www.emdbiosciences.com/docs/docs/LIT/Complement_CB0617_EUSD.pdf
2 Weiszhár Z, Czúcz J, Révész C, Rosivall L, Szebeni J, Rozsnyay Z. Complement
activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, Tween-80
and Tween-20. Eur J Pharm Sci. 2012 Mar 12;45(4):492-8.
3 Szebeni J, Muggia F, Gabizon A, Barenholz Y. Activation of complement by
therapeutic liposomes and other lipid excipient-based therapeutic products:
prediction and prevention. Adv Drug Deliv Rev. 2011 Sep 16;63(12):1020-30
4. Dobrovolskaia MA, McNeil SE. Understanding the correlation between in vitro
and in vivo immunotoxicity tests for nanomedicines. J Control Release.
2013;172(2):456-66.
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5. Chanan-Khan A, Szebeni J, Savay S, Liebes L, Rafique NM, Alving CR, Muggia
FM. Complement activation following first exposure to pegylated liposomal
doxorubicin (Doxil): possible role in hypersensitivity reactions. Ann Oncol. 2003,
14, 1430-1437.
13. Abbreviations
CVF cobra venom factor
PBS phosphate buffered saline
EIA enzyme immunoassay
HAGG heat aggregated gamma globulin
HRP horseradish peroxidase
IEC inhibition/enhancement control
IgG (H + L) immunoglobulin G (high and low chains)
NC negative control
PC positive control
